After acquiring a microscopic image, the resulting digital image is generally processed. The final outcome of the processing procedure can be either an improve image, or a set of parameters obtained from careful analysis and treatment of the image. The PPBI consortium has several image analysis & processing software either licensed or freeware that will help the analysis of the obtained data, as well as greatly enhance the final image. Among the licensed software available there are Huygens (image restoration, visualization, and analysis); Imaris (data visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets); MatLab (analyze data, develop algorithms, create models and applications); IN Cell Investigator (flexible and comprehensive solution for automated high-content analysis of live and fixed assays). Among the freeware software there is Image J (display, edit, analyze, process, save and print 8-bit, 16-bit and 32-bit images), Fiji (an image processing package. It can be described as a "batteries-included" distribution of ImageJ); Cell Profiler (open-source platform for automated image analysis) Cell Profiler Analyst (open-source software for exploring and analyzing large, high-dimensional image-derived data), GNU Octave (high-level language, primarily intended for numerical computation)

High-throughput microscopy and "high content screening" allow the automated acquisition and analysis of large numbers of cells, which may also include automatic detection and live-tracking of specific cell phenotypes/behaviours. HTM systems are available currently at IBMC, FCUL and IGC nodes, but lab support for HCS analysis expertise exists mostly at the IBMC and FCUL nodes.

Though PPBI entails mostly facilities which specialize in optical imaging, some nodes do provide access to electron microscopy resources especially (but not limited to) projects which involve some form of correlative optical and electron microscopy (techniques generally known as CLEM - Correlative Light and Electron Microscopy, or 3D reconstruction of biological samples at EM resolution using "3D electron tomography". This possibility exists at nodes at IBMC, FCUL and IGC.

PPBI comprises multiple light microscopy resources including high-end widefield microscopes, laser scanning and spinning disk confocal systems, two-photon microscopes and super-resolution. TECHNIQUES AVAILABLE: Multichannel (widefield) fluorescence imaging + transmission imaging • 3D imaging and reconstruction with laser scanning or spining disk confocals, but may also have multiphoton (2P) or light-sheet (LSM) • Multidimensional in vivo timelapse experiments (ie, equipped with incubators and environmental control) • Mosaic imaging (imaging of large areas with multiple fields and stitching of adjacent fields; eg large tissue sections or whole embryos/organs) • High throughput microscopy; typically fully automated scope with protocols for acquiring automatically multiple fields in multi-well plates or dishes • Fluorescence Recovery After Photobleaching (FRAP), Photoactivation (PA) and Photoconversion (PC) • Fluorescence Resonance Energy Transfer (FRET) • Fluorescence-Lifetime Imaging Microscopy (FLIM) and Fluorescence Correlation Spectroscopy (FCS) • High-speed live cell microscopy and analysis (typicaly acquisition of multiple images /second) • "Super Resolution" and Single Mol. Localization techniques

Mesoscopy is the whole imaging (3D) or large samples ranging from 1mm to a few cm, with techniques such as light-sheet microscopy (aka SPIM) or optical (projection) tomography (aka OPT), among others. SPIM is well suited for live imaging of small embryos such as zebrafish or drosophila, less than 2mm and ~500um thick. For larger samples it is advisable to use OPT, however, in this case imaging is limited to fixed samples. It is also possible to image fluorescence and bioluminescence in vivo using animal imagers, and have those images registered with X-ray images of the whole animal. Mesoscopy resources are available at INEB and IGC nodes.